Although our current knowledge about mammalian DNA replication suggests that many enzymes are involved, none have until now been shown to be required. To determine which enzymes are required for DNA replication, I will analyze the replication in vitro of Simian virus 40 (SV40) chromosomes. Replicating SV40 chromosomes capable of continuing DNA synthesis can be isolated; we will fractionate the enzymes bound to these chromosomes and examine their role(s) in DNA replication. For each enzyme detected, we will determine whether it is preferentially associated with replicating SV40 chromosomes. We will also examine whether its removal or inhibition results in an alteration in the process of DNA replication; such an alteration provides excellent evidence for the role(s) of that enzyme. If no effect is found, the possibility of adventitious association will be tested. A pilot study, described below, has shown the great potential of this approach. We have demonstrated that one of the three mammalian DNA polymerases, DNA polymerase alpha, performs virtually all of the SV40 DNA synthesis observed. The study proposed here should allow us to determine what other enzymes are involved in DNA replication. Knowledge of the enzymes involved in replication will allow comparisons between normal and malignant cells, to attempt to understand the uncontrolled cell proliferation of the latter. It would also improve our understanding of the mechanism of action of chemotherapeutic and antiviral agents directed at DNA replication, and provide a rational basis for design of new drugs that attack specific replication enzymes. In addition, the characterization of this in vitro DNA replication system should make it an excellent model to probe the effects of carcinogens on the process of DNA replication.